Cancer genomic profiling identified dihydropyrimidine dehydrogenase deficiency in bladder cancer promotes sensitivity to gemcitabine

Chemotherapy is a standard therapy for muscle-invasive bladder cancer (MIBC). However, genomic alterations associated with chemotherapy sensitivity in MIBC have not been fully explored. This study aimed to investigate the genomic landscape of MIBC in association with the response to chemotherapy and to explore the biological role of genomic alterations. Genomic alterations in MIBC were sequenced by targeted exome sequencing of 409 genes. Gene expression in MIBC tissues was analyzed by western blotting, immunohistochemistry, and RNA microarray. Cellular sensitivity to gemcitabine and gemcitabine metabolite was examined in bladder cancer cells after modulation of candidate gene. Targeted exome sequencing in 20 cases with MIBC revealed various genomic alterations including pathogenic missense mutation of DPYD gene encoding dihydropyrimidine dehydrogenase (DPD). Conversely, high DPYD and DPD expression were associated with poor response to gemcitabine-containing chemotherapy among patients with MIBC, as well as gemcitabine resistance in bladder cancer cells. DPD suppression rendered cells sensitive to gemcitabine, while DPD overexpression made cells gemcitabine-resistant through reduced activity of the cytotoxic gemcitabine metabolite difluorodeoxycytidine diphosphate. This study revealed the novel role of DPD in gemcitabine metabolism. It has been suggested that DPYD genomic alterations and DPD expression are potential predictive biomarkers in gemcitabine treatment.

benefit from the chemotherapy, while the other 60% are at risk of complications and delay in definitive surgical intervention without clear benefit 1 . It is therefore critical to predict NAC efficacy in patients with MIBC in order to administer NAC more selectively prior to radical cystectomy 4 . Predictive biomarkers of response to chemotherapy would have significant impact on personalized multimodal therapy in patients with MIBC.
Thus far, several studies have demonstrated the association between specific genomic alterations and tumor response to chemotherapy. In urothelial carcinoma including BC, somatic mutations in DNA damage repair genes (ERCC2, ATM, RB1, FANCC, and BRCA2) have been correlated with sensitivity to cisplatin 1,[7][8][9] . However, analyses beyond DNA damage repair genes to predict tumor response to chemotherapy in BC has been limited. Therefore, this study aimed to reveal genomic alterations associated with tumor response to platinum-based chemotherapy for BC by targeted exome sequencing of 409 genes. On the basis of the findings obtained by targeted exome sequencing, the biological role and clinical value of identified genomic alterations were explored.

Results
Mutation landscape in MIBC by targeted exome sequencing. To characterize the genomic landscape of MIBC, we performed targeted exome sequencing of 409 cancer-associated genes in MIBC tissues from 20 patients. The clinicopathological characteristics are provided in Supplementary Table S1, and their clinical courses are shown in Fig. 1a. Among 20 patients, 13 received platinum-based chemotherapy. Tumor response to chemotherapy was good (ypT0N0 after NAC or complete response during primary/salvage treatment) in 4 patients, and poor (pN1 after NAC or stable disease or progressive disease after primary/salvage treatment) in 9 patients. During a median follow-up of 14.7 months (interquartile range, 12.2-19.6 months), 11 patients developed metastasis, and seven patients died due to disease progression among 20 patients with MIBC. Notably, five patients among nine poor responders died from BC (Fig. 1a). By targeted exome sequencing, 1159 somatic mutations in total were detected in the tumor tissue from 20 patients (Fig. 1b, Supplementary Table S2). Sequencing of leukocyte-derived gDNA has confirmed that gene mutations are not germline mutations. Tumor mutation burden (TMB) was comparable among tumor tissues with good (25.2 muts/Mb) and poor (21.3 muts/ Mb) responses (P = 0.594).
All genomic alterations were classified as non-pathogenic mutation, and pathogenic mutation. The following genomic alterations reported to be associated with response of chemotherapy were detected; TP53 in 6 cases, ERCC2 in 5 cases, FANCC in 1 case, ATM in 4 cases, and RB1 in 3 cases ( Fig. 1b and Supplementary Fig. S1a). Although the frequency of genomic alterations in most genes differed from those reported in TCGA study, the frequency of DPYD alteration was comparable with that reported in TCGA study ( Fig. 1c and Supplementary  Fig. S1b).
Interestingly, patient #04 harbored a single-nucleotide variant (c.1031 C > T) leading to amino-acid substitution as p.Ala334Val (valiant allele frequency, 48.5%) (Fig. 2a), and showed a complete response of lung metastasis and the primary bladder tumor to 6 cycles of GC chemotherapy followed by cystectomy (Fig. 2b). This missense mutation in DPYD gene was judged as pathogenic by Polyphen2 (score = 1.000). Then, among various gene alterations, we focused DPYD gene encoding dihydropyrimidine dehydrogenase (DPD) because gemcitabine metabolite structure, difluorodeoxyuridine (dFdU), was similar to 5-FU, to which DPD is well known to be involved in cellular resistance 10,11 . DPD expression was associated with response to chemotherapy. DPD plays a critical role in the metabolism of pyrimidine-based drugs including gemcitabine 12 , and therefore we focused on the clinical significance of DPD in gemcitabine-containing chemotherapy. First, the association between DPD expression and response to chemotherapy was examined in tumors with good and poor responses to chemotherapy. DPD protein expression was lower in tumors with a good response while DPD protein expression was higher in tumors with a poor response (P = 0.012, Fig. 3a, Supplementary Fig. S2a and S2b). The sensitivity and specificity of predicting response from DPD protein expression at a cutoff value of 0.804 were 0.857 and 1.000, respectively (Fig. 3b). Consistently, representative imaging obtained by immunohistochemistry showed an absence of DPD staining in tissues from the good responders, while positive DPD staining was detected in tissues from the poor responders (Fig. 3c).
Next, the association between DPYD mRNA expression and tumor response to NAC was explored using microarray data (GSE87304) from 223 patients who received NAC regimens including gemcitabine 13 . When the association between DPYD mRNA expression and molecular subtypes, DPYD expressions were highest and lowest in claudin-low and luminal subtypes, respectively (Fig. 3d). We separated 169 patients who received gemcitabine-containing NAC from 54 patients who received non-gemcitabine containing MVAC, and compared the highest and lowest quartiles in each treatment group based on DPYD expression (Table 1). Residual extravesical extension after gemcitabine-containing NAC (ypT3/4) was found more frequently among tumors with high DPYD expression, compared with tumors with low DPYD expression (P = 0.019, Fig. 3e). Overall survival was worse in patients with DPYD-expressing tumors compared to those with low DPYD-expressing tumors after treatment with gemcitabine-containing NAC (Fig. 3f). In contrast, DPYD expression was associated with neither tumor response nor overall survival in patients treated with MVAC (Supplementary Figs. S3a and S3b). Similarly, TCGA study showed that disease-free survival was comparable between higher and lower expression of DPYD among 137 patients without chemotherapy ( Supplementary Fig. S3c) 14 .
GSEA analysis was performed to ascertain the gene networks associated with DPYD expression using the cohort (GSE87304). Although several gene set were significant, there was no apparent functional signature associated with gemcitabine resistance (Supplementary Table S3). www.nature.com/scientificreports/ www.nature.com/scientificreports/ DPD expression modulated cellular sensitivity to gemcitabine. To delineate the association between DPD expression and sensitivity to gemcitabine, DPD expression was modulated and cellular sensitivity to gemcitabine was examined. When DPD expression was reduced using specific siRNA, UM-UC-3 cells became sensitive to gemcitabine (Fig. 4a, Supplementary Fig. S4a and S4d), but not to cisplatin ( Supplementary Fig. S5). Conversely, stable DPYD overexpression using a DPYD-expression plasmid conferred resistance to gemcitabine in HEK293 cells ( Fig. 4b and Supplementary Fig. S4e-S4k). However, when mutated DPYD (c.1031C > T) found in patient #04 was overexpressed in HEK293 cells, cellular viability was lower compared with cells overexpressing wild-type DPYD ( Fig. 4c and Supplementary Fig. S4l-S4r). Next, DPYD mRNA and DPD protein expression was compared between parental-(P) and gemcitabineresistant (GEM-R) cell lines. Higher expression levels of DPYD mRNA and DPD protein were observed in RT112 GEM-R and UM-UC-13 GEM-R cells compared with parental cells (Fig. 4d and Supplementary Fig. S4s-S4u). When DPD protein expression was reduced using specific siRNA in RT112 GEM-R cells, gemcitabine resistance was reversed (Fig. 4e, Supplementary Fig. S4v and S4x).
Gemcitabine metabolism by DPD. Finally, we investigated the mechanism of DPD-derived gemcitabine resistance by examining nucleotide metabolism with LC-MS/MS. Difluorodeoxycytidine diphosphate (dFd-CDP, a gemcitabine metabolite) is a ribonucleotide reductase (RNR) inhibition. RNR is an enzyme that converts ribose to deoxyribose, which is very important for generating dNTPs for DNA synthesis. As a result of the inhibition of RNR, increased intracellular concentrations of NMP, NDP, and NTP can be observed as an effect of gemcitabine because deoxyribonucleic acid is depleted while ribonucleic acid is stored. As shown in Fig. 5, gemcitabine induced increased levels of ribonucleotides and ribonucleosides in low DPD-expressing HEK293 cells. In contrast, these metabolic changes were abrogated in DPYD-overexpressing HEK293 cells (Fig. 5).

Discussion
Clinical data in this study suggested that genomic alteration in DPYP and gene expression level of DPYP were associated with response to gemcitabine-containing chemotherapy. In line with this notion, experimental data in this study have shown that DPD promoted resistance to gemcitabine through deactivation of gemcitabine. www.nature.com/scientificreports/ DPD expression is associated with tumor response to 5-fluorouracil (5-FU) in BC cells [10][11][12][13][14][15][16][17] . Because 5-FU is metabolized and deactivated by DPD, DPD may protect cells from cytotoxicity by 5-FU ( Supplementary  Fig. S6) 10 . Similarly, this study revealed that DPD is a critical determinant of chemosensitivity to gemcitabine in BC. Gemcitabine (difluorodeoxycytidine; dFdC) is a pyrimidine antagonist, similar to 5-FU. As a molecular mechanism involved in gemcitabine resistance, gemcitabine metabolism and deactivation by DPD were suggested. Gemcitabine is metabolized to the bioactive form of difluorodeoxycytidine monophosphate (dFdCMP), diphosphate (dFdCDP, a potent inhibitor of the enzyme ribonucleotide reductase), and triphosphate (dFdCTP, incorporated as a false precursor) by dCK, nucleotide monophosphate kinase (NMPK), and nucleotide diphosphokinase (NDPK), respectively ( Supplementary Fig. S6). Meanwhile, more than 90% of gemcitabine is inactivated by CDA into dFdU 10 , which may be a substrate of DPD and metabolized by DPD ( Supplementary Fig. S6). Then, we hypothesized that impaired activity of DPD by low expression or dysfunction due to mutations may result in decrease inactivation of gemcitabine, and instead increased metabolism of gemcitabine to its bioactive form. Actually, for the first time, to our knowledge, this study showed that DPD robustly modulated nucleotide metabolism, suggesting deactivation of gemcitabine by DPD.
Previous study has reported that DPD expression is associated with the clinical benefit of adjuvant 5-FU chemotherapy for upper urinary tract urothelial carcinoma 16 . Furthermore, in the ESPAC-3(v2) clinical trial, high expression of DPD was associated with poor survival when patients were treated with 5-FU or gemcitabine for pancreatic cancer 18 . Consistently, this study showed that high DPYD expression was associated with poor response and survival following administration of gemcitabine-containing chemotherapy. Additionally, in our analysis of MIBC microarray data, high and low DPYD expression was associated with poor-prognosis claudinlow and good-prognosis luminal subtypes, respectively. Thus, the molecular subtypes may be associated with prognosis through DPD expression in MIBC.
Genetic variants in DPYD were reported to affect 5-FU metabolism and are associated with adverse events in chemotherapy using gemcitabine for pancreatic cancer 19,20 . However, this study notably reported the significance of DPYD genomic alterations of tumor cells following gemcitabine-containing chemotherapy. Thus, while germline deleterious loss-of-function in DPYD results in increased cytotoxicity by gemcitabine in normal cells, somatic alterations in cancer cells lead to augmented clinical benefit by gemcitabine. Taken together, these findings indicated that DPYD mRNA expression and DPD protein expression, as well as genomic alterations in DPYD, in cancer cells may be predictive markers of efficacy in 5-FU-and gemcitabine-containing chemotherapy regimens.
However, this study suffers from some limitations. Sample number for analysis of genomic alterations by targeted exome sequencing was small to perform statistical analysis. In addition, there was imbalance of sample number between good and poor responders. HEK293 and UM-UC-3 cells were utilized in in vitro experiments. However, HEK293 cells were not BC cell line, and UM-UC-3 cells are a hypertriploid cell line, which might affect the result in this study (CRL-1749, ATCC, https:// www. atcc. org/ produ cts/ crl-1749).
In conclusion, this study demonstrated the genomic landscape of MIBC, and the novel role of DPD in gemcitabine metabolism, suggesting that DPD is a critical molecule in cellular sensitivity to gemcitabine through gemcitabine deactivation. DPYD genomic alterations and expression levels may be useful biomarkers to predict tumor response to gemcitabine-containing chemotherapy in BC.

Materials and methods
Sample collection. This study included 20 patients diagnosed with MIBC at Kyushu University Hospital (Fukuoka, Japan) between 2019 and 2021. Of these 20 patients, nine and four patients received chemotherapy as neoadjuvant and primary/salvage treatments, respectively. Tumor tissues and blood samples were banked at − 80 °C until extraction of DNA or protein. Written informed consent was obtained from all patients, and the study was approved by the institutional review board (#2020-254). The study was conducted in accordance with the principles described in the Declaration of Helsinki and the Ethical Guidelines for Epidemiological Research enacted by the Japanese Government.
Targeted exome sequencing. Tumor DNA was extracted from tissues obtained by transurethral resection (TUR) or radical cystectomy with a DNeasy blood and tissue kit (Qiagen, Hilden, Germany). Germline DNA (gDNA) was extracted from blood collected before undergoing chemotherapy. Targeted exome sequencing was performed using the Ion Ampliseq Comprehensive Cancer Panel (CCP; #4,477,685; Thermo Fisher, Waltham, MA, USA), which consisted of four primer pools totaling nearly 16,000 primer pairs covering 1.23 Mb of 409 genes (Supplementary Table S4) 21 . DNA (10 ng) from 20 tumors and matched gDNA was used for libraries. Libraries were generated by the Ion Torrent Ion Chef system (Thermo Fisher) with the Ion Ampliseq Library kit plus (Thermo Fisher) and sequenced by an Ion PGM Sequencer (Thermo Fisher). We analyzed sequences using Ion Reporter Software 5.18.1.0 (Thermo Fisher, https:// ionre porter. therm ofish er. com/ ir/). The mean depth of read coverage for the target genes was 10 3 × (interquartile range, 48.5-139). Mutations reported as "pathogenic" or "likely pathogenic" in dbSNP (https:// www. ncbi. nlm. nih. gov/ snp/), and mutations with Polyphen2 score > 0.9 (http:// genet ics. bwh. harva rd. edu/ pph2/) were determined to be pathogenic 22 . A mutation was defined as having a 5.0% allele frequency or greater, and TMB was calculated by dividing the number of mutations by the sequence length of the comprehensive cancer panel (1.23 Mb). Public dataset of the TCGA study (BLCA) were obtained from cBioPortal (www. cbiop ortal. org), and the frequency of mutation in TP53, ATM, ERCC , RB1, DPYD, FANCC genes was analyzed 23,24 . DYPD mRNA expression analysis in MIBC tissues. Microarray data (GSE87304) from a multi-institutional cohort of 223 MIBC patients treated with NAC followed by radical cystectomy were used 13 25 . Public dataset of the TCGA study (BLCA) were obtained from cBioPortal (www. cbiop ortal. org), and the gene expression of DPYD was analyzed 23,24 .    (Invitrogen), respectively, in accordance with the manufacturer's instructions 28 . Stable overexpressing cells were established by transfecting expression plasmid into HEK293 cells as described above, and culturing with medium containing G418 (Nacalai tesque) at 400 μg/mL for at least 2 weeks for selection.

Cell proliferation assay. Cell proliferation was calculated by cell counting or CellTiter-Glo Luminescent
Cell Viability Assay (Promega, Madison, WI, USA). Cell counting was performed using a TC20 Automated Cell Counter (Bio-Rad, Hercules, CA, USA) after incubation in the indicated conditions. For CellTiter-Glo assay, cells were seeded in a 96-well plate with phenol-red free medium and cultured under the indicated conditions. CellTiter reagents were added to the medium and incubated for 10 min, followed by measurement using a 2030 ARVO X2 (PerkinElmer, Waltham, MA, USA) 29 . Cell survival rate when exposed to gemcitabine was corrected with that in the absence of gemcitabine. The proliferation ratio was calculated using the formula below. A proliferation ratio of > 1.0 indicated resistance and that of < 1.0 indicated sensitivity.  www.nature.com/scientificreports/ Figure 5. Nucleic acid-related metabolites after exposure to gemcitabine. Nucleic acid-related metabolites in HEK293 cells and DPD-overexpressing HEK293 cells cultured in medium containing 0 or 6.67 nM gemcitabine for 24 h. The concentrations of metabolites when exposed to gemcitabine were corrected against those not exposed to gemcitabine. Boxes, mean; bars, ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t-test).  31  Plasma metabolite preparation. Metabolite extraction from cultured cells was described previously 32 .
To prepare nucleic acid-related metabolites, approximately 3 × 10 6 HEK293 cell pellets were added to 500 µL ice-cold 80% methanol, vortexed, sonicated five times (30-s sonication and 30-s cooling) with a BIORUPTOR (Cosmo Bio, Tokyo, Japan), and centrifuged at 21,500×g for 5 min at 4 °C. The supernatants were collected, and 250 µL supernatant was evaporated to dryness, then dissolved in a mobile phase determined by each analytical condition and then subjected to LCMS measurement of nucleotides or nucleosides.
Statistical analysis. All statistical analyses were performed using EZR software (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interface for R 2.13.0 34 . Statistical analyses were performed using Fisher test, Student's t-test, one-way ANOVA, and Mann-Whitney U test. All P-values are twosided. Levels of statistical significance were set at P < 0.05.

Ethical compliance. The study was approved by Kyushu University Institutional Review Board for Clinical
Research (#2020-254).

Data availability
The authors declare that the sequencing data of 20 patients of this study are available within the paper and its Supplementary Information files. The microarray data that support the findings of this study are available from University of British Columbia, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of University